UPTAKE OF ATRIAL-NATRIURETIC-PEPTIDE AND PRODUCTION OF CGMP IN CULTURED HUMAN GLOMERULAR ENDOTHELIAL-CELLS

Interactions between human glomerular endothelial cells and atrial nat riuretic peptide (ANP) were studied with I-125-alpha-human-ANP binding and intracellular accumulation of cGMP. Uptake for alpha-hANP (1-28 o r 5-28) by homogeneous cultures of human glomerular endothelial cells was dose and time dependent with optimal uptake occurring after 30 min of incubation at 37 degrees C. Scatchard analysis of the specific bin ding data with a two-compartment model identified both high (K-d, = 0. 3 nM)- and low (K-d = 10 nM)-affinity receptors, with a binding site d ensity of 12,000 and 18,060 receptors per cell, respectively, alpha-hA NP markedly stimulated glomerular endothelial cell-associated cGMP. Af ter a 2-min incubation, cGMP increased 1.3-fold (from 17.88 +/- 1.29 t o 23.33 +/- 3 pmol/mg of protein), in the presence of 1 nM ANP, to mor e than threefold (from 21 +/- .1 to 80.5 +/- 14.5 pmol/mg of protein) with 1 mu M ANP (P < 0.05). In contrast, a 10 mu M concentration of th e clearance receptor C-ANP(4-23) increased cGMP by 1.6 +/- 0.6 fold. A NP stimulation of intracellular cGMP was 100 times more sensitive in h uman glomerular endothelial than in mesangial cells. In comparison, hi gher doses of bradykinin were necessary to evoke similar responses in glomerular endothelial cells. In the presence of 10 mu M bradykinin, c ellular cGMP increased by 1.75 +/- 0.6-fold versus control cells. Howe ver, unlike ANP, bradykinin-stimulated cGMP synthesis was significantl y inhibited by prior treatment with oxyhemoglobin (10(-5) M), an inhib itor of soluble guanylate cyclase, and N-G-nitro-L-arginine (NO(2)Arg) , a specific inhibitor of endothelial-derived relaxing factor (EDRF). ANP-stimulated cGMP synthesis was also attenuated in the presence of b oth hemoglobin and NO(2)Arg; however, the difference was not significa nt compared with ANP alone. In addition, alpha-hANP had no effect on i ntracellular free calcium or the accumulation of cAMP. In summary, hum an glomerular endothelial cells possess both high- and low-affinity bi nding sites for ANP, which respond by the activation of guanylate cycl ase. The attenuation of ANP-stimulated cGMP synthesis in vitro by spec ific blockers of EDRF suggests that locally released EDRF may modulate the cellular effects of ANP.